Group B Streptococcus (GBS), a causative agent of newborn infection, colonizes the digestive system and genitourinary tracts of pregnant women. The aim of this study was to assess the incidence of GBS capsular serotypes, virulence genes, and antibiotic resistance in pregnant women at a prominent maternity hospital in Shahrekord, Chaharmahal and Bakhtiari. From January to July 2022, a total of 370 vaginal swabs were collected from women who were from 28 to 38 weeks pregnant. These swabs were used to identify Streptococcus agalactiae using PCR and culture technique. The capsular serotype of Streptococcus agalactiae isolates, antibiotic resistance and virulence genes distribution were determined through disk diffusion and multiplex PCR methods. mong 370 vaginal samples, 36 (9.72%) isolates were detected by culture and molecular methods, that type III (37.83%) and type V (32.43%) were the most frequent capsular serotypes, and all isolates harbored atr, and dltS genes. Moreover, the most frequent antibiotic resistance genes (62.16%) in the isolates is was related to tetO and ermA. All isolates were resistant to penicillin (100%), and the highest antibiotic susceptibility was found against linezolid (81%). Our study revealed a high rate of antibiotic resistance in Streptococcus agalactiae isolates, that molecular method was reliable approach for identification of infection and detection of harbored resistance genes. However, more studies are required to identification of effective antibiotics against infections and prevention of resistance in pregnant women.